Trypanosoma cruzi is a protozoan parasite that causes a fatal illness called Chagas' disease or American typanosomiasis in humans and many nonhuman mammalian species. The World Health Organization has estimated that 18 million people are infected with T. cruzi and that 50,000 patients die each year of the disease. The basic research on T. cruzi over the past two decades has resulted in many advances in our understanding of these organisms, yet these substantial research efforts have not led to a better way to manage or control this devastating disease. Recently, progress on the human genome project, and the determination of the yeast genomic sequence, have unambiguously demonstrated the value of using genomic sequences as a foundation for designing future research efforts. Our long term goal is to sequence the genome of T. cruzi. This proposal, in conjunction with similar proposed efforts at the Seattle Biomedical Research Institute and Uppsala University, represents a large step towards that goal. We will employ a sequencing strategy consisting of two phases. The purpose of the first phase is to enhance early gene discovery and to provide markers that will be important for construction of a high-resolution sequence-ready map. In this phase we will generate approximately 10 Mb of discontinuous single-pass sequence (23 percent of the 43.5 Mb genome). This will be implemented by end-sequencing 5,000 BAC (Bacterial Artificial Chromosome) clones from an already existing library and another 5,000 BAC clones of randomly-sheared DNA (from a library to be constructed in collaboration with Dr. Pieter deJong). Telomere-proximal sequences will be identified in clones of the sheared genonmic library. During the second phase 12 Mb of contiguous T. cruzi DNA will be sequenced using a chromosome by chromosome approach and a syntenic regions conserved between T. cruzi and a related parasite (Trypanosoma brucei) will be identified and analyzed. This project will provide invaluable information and benefits at many levels, including (1) identification of genes involved in basic functions of the eukaryotic cell, (2) easy, inexpensive and fast cloning of genes encoding proteins being actively studied in laboratories around the world, (3) immediate access to genes and their products for functional/structural studies, (4) prediction of metabolic pathways on the basis of candidate genes, (4) identification of parasite-specific gene products by comparison with other genomes, and (5) a framework for future experiments aimed at comparative and integrative mapping of various trypanosomatid genomes.